Geranylgeranylacetone selectively binds to the HSP70 of Helicobacter pylori and alters its coccoid morphology

Abstract

Geranylgeranylacetone (GGA) is used to treat patients suffering from peptic ulcers and gastritis. We examined the effect of GGA on Helicobacter pylori, which is a causative factor of gastrointestinal diseases. Previously, we have reported that GGA binds specifically to the molecular chaperone HSP70. In this paper, we report that GGA bounds to H. pylori HSP70 (product of the DnaK gene) with 26-times higher affinity than to human HSP70, and induced large conformational changes as observed from surface plasmon resonance and circular dichroism. Binding of GGA suppressed the activity of the H. pylori chaperone. GGA also altered several characteristics of H. pylori cells. GGA-treated cells elicited enhanced interleukin-8 production by gastric cancer cell lines and potentiated susceptibility to complement as compared to untreated cells. GGA also caused morphological alterations in H. pylori as reflected in fewer coccoid-like cells, suggesting that GGA converts H. pylori to an actively dividing, spiral state (vegetative form) from a non-growing, coccoid state. This morphological conversion by GGA resulted in accelerated growth of H. pylori. These results suggest a model in which GGA sensitizes H. pylori to antibiotic treatment by converting the cells to an actively growing state.

Figure 1. Purified DnaK and HSP70.

DnaK and HSP70 were purified as described under “Materials and Methods” and the purity was analyzed by SDS-PAGE (9% gels).

Figure 2. Surface plasmon resonance analysis of the interaction between HSP70 or DnaK and GGA.

A sensorgram for the binding of DnaK (A) or HSP70 (B) to GGA is shown. Different concentrations of GGA were injected as described under “Materials and Methods”. RU, resonance units.

Figure 3. Effect of GGA on the conformational changes of DnaK and HSP70.

(A) the CD spectrum of DnaK was measured in the absence (closed red diamond) or presence (closed blue square) of GGA as described under “Materials and Methods” denotes the mean residue ellipticity. (B) the CD spectrum of HSP70 was measured in the absence (closed red diamond) or presence (closed blue square) of GGA as described under “Materials and Methods” the mean residue ellipticity.

Figure 4. Effect of GGA on the chaperone activity of DnaK and HSP70.

(A) time course of 50 μM DnaK (closed black diamond), 0.25 μM citrate synthase (CS) aggregation in buffer (closed red square), buffer containing 50 μM DnaK (closed yellow triangle), buffer containing 50 μM DnaK and 0.5 mM GGA (closed purple circle), buffer containing 50 μM DnaK and 2.5 mM GGA (closed blue circle), and buffer containing 50 μM DnaK and 5.0 mM GGA (closed green circle). (B) time course of 50 μM HSP70 (closed black diamond), 0.5 M citrate synthase aggregation in buffer (closed red square), buffer containing 50 μM HSP70 (closed yellow triangle), buffer containing 50 μM HSP70 and 0.5 mM GGA (closed purple circle), buffer containing 50 μM HSP70 and 2.5 mM GGA (closed blue circle), and buffer containing 50 μM HSP70 and 5.0 mM GGA (closed green circle).

Figure 5. Effect of GGA on H. pylori cell growth.

GGA was added to H. pylori SS1 culture in BHI-FBS broth at 37 ^oC under microaerophilic condition at a concentration of 0 (closed black diamond), 0.2 (closed red square), 1 (closed purple triangle), or 5 (closed blue circle) mM. The cells were cultured, and turbidity (determined by A₆₀₀) was measured.

Figure 6. Effect of GGA on H. pylori cell morphology.

H. pylori SS1 was cultured in BHI-FBS broth at 37 ^oC under microaerophilic condition in the absence or presence of GGA at a concentration of 0 (A), 1 (B), and 5 mM (C). Bacterial smear was stained by Gram staining. Coccoid form cells (D) were prepared by cultivation under anaerobic condition. Arrows in (A) indicate coccoid-like cells.

Figure 7. Effect of GGA on serum sensitivity of H. pylori cells.

H. pylori SS1 was precultured in the absence or presence of GGA at a concentration of 0 (open circle), 1 (closed red diamond), and 5 (closed black square) mM. The resulting cells were treated with 50% normal rabbit serum as a source of complement. After incubation at various times, the suspension was diluted appropriately and plated on sheep blood agar plates. After 72 h culture, the colonies were counted. Each experiment was performed in quadruplicate.

Figure 8. Effect of GGA on Interleukin-8 (IL-8) production induced by H. pylori live cells in gastric carcinoma cell line MKN28 and MKN45.

H. pylori SS1 was precultured in the absence or presence of GGA at a concentration of 0 (open circle), 1 (closed red diamond), and 5 (closed black square) mM. The resulting cells were inoculated to MKN28 cells or MKN45 cells at MOI 10 or 100. After 24 h incubation, IL-8 in the culture supernatant was measured by ELISA. Each experiment was performed in triplicate.